Id proteins are master regulators of cell proliferation, differentiation and self-renewal of stem cells. Development of the adult organism requires suppression of Id function whereas persistent Id activation leads to tumor formation. Aberrant expression of Id proteins has been detected in most types of human cancer, in which it sustains key hallmarks of tumor progression such as unrestrained proliferation, anaplasia and tumor angiogenesis. During the past funding cycle, we demonstrated that the proliferative and angiogenic responses induced by Id are rate limiting for tumor development. Using a mouse tumor model in which pituitary cancer is initiated by loss of the Rb tumor suppressor gene, we discovered that each of the crucial traits of tumor growth (initiation, tumor cell proliferation and angiogenesis) requires Id activity. Collectively, these findings suggest that targeting Id proteins may have widespread benefit in preventing tumor initiation and maintenance. All analyses described above were performed using tissues and cells from Id2 knockout mice in which the Id2 gene had been constitutively inactivated throughout embryogenesis. What is the requirement for Id function after the acute and combined deletion of all the Id genes? The embryonic lethality of compound Id null mice precluded us from addressing this question. We are in the process of generating conditional Id1/2/3 triple-knockout mice. In Aim 1, we will use cells and tissues from these mice to by-pass the embryonic lethality and study Id function in the neural stem cell compartment. We previously showed that Id2-null cells showed greatly reduced susceptibility to oncogenic transformation. A critical, unresolved question is whether ablation of Id genes in already transformed cells would revert the transformed phenotype. Given the pervasive and aggressive nature of the angiogenic switch in neuroectodermal tumors and its dependency on Id expression, we have focused on this cellular system. Thus, we will address this issue in Aim 2 by testing the requirement for Id function in a mouse model for malignant glioma. This issue is very important from a clinical standpoint, and it must be resolved before anti-Id therapy in human cancer patients is entertained. We found that Id proteins enhance tumor angiogenesis by increasing the production of Vascular Endothelial Growth Factor (VEGF) through stabilization of the Hypoxia Inducible Factor alpha (HIF?), the main inducer of VEGF expression in human cancer. We have begun exploring the mechanistic basis of this observation. We have now found that Id proteins interfere with the assembly of the Von Hippel Lindau (VHL) ubiquitin ligase, a multi- subunit tumor suppressor complex whose primary function is destruction of HIF? in oxygenated cells. In the work described in Aim 3, we will follow up on these observations with detailed mechanistic analyses. Thus, the proposed study for the next funding cycle will conclusively address the role of Id proteins in the initiation and progression of malignant brain tumors and validate Id as targets for therapeutic intervention in this incurable cancer. PUBLIC HEALTH RELEVANCE: Id proteins are master regulators of cell proliferation, differentiation and self-renewal of stem cells. Development of the adult organism requires suppression of Id function whereas persistent Id activation leads to tumor formation. Malignant gliomas are among the most lethal human cancers. In this proposal, we will study how Id proteins promote relentless tumor cell proliferation and angiogenesis, key hallmarks of tumor progression in the brain. Ultimate goal of our work is to provide novel therapeutic targets to attack this intractable disease.